If a virus is to be used in vaccine challenge studies, it is essential to know its minimum infectious dose. In this project, the highly pathogenic lentivirus, HIV-2287, was titered in vivo with inoculation by either rectal or vaginal mucosa routes. In the first part of this experiment, 14 macaques were assigned as follows intravaginal (IVg) inoculation six female juveniles, one pair each at doses of 104, 103, and 102 TCID; intrarectal (IR) inoculation three male and three female juveniles, one of each at the same doses as above; intravenous control inoculation two macaques, one each at doses of 100 and 10 TCID. Previous intravenous titrations have shown that 1 TCID is ~1 animal infectious dose (AID). The macaques inoculated at the lowest mucosal doses, whether IVg or IR, showed levels of infection that could only be detected by PCR. At doses of 104 or 103, all macaques became infected as shown by co-culture, and most developed CD4+ cell depletion. Both the control macaques became infected, at levels proportional to the dose. In a subsequent experiment to refine the minimum dose, two macaques, one male and one female, were inoculated intrarectally with 500 TCID. This dose produced infection and CD4+ cell depletion in one of the two macaques, indicating that the minimum infectious dose by intrarectal inoculation is between 500 and 1000 TCID. Two adult female macaques were also inoculated with 103 TCID intravaginally. Unlike the case in juvenile macaques, this dose did not produce the same level of infection in the adult macaques. Additional experiments are underway to examine the impact of reproductive cycling in the adult female on the minimum infectious dose of HIV-2287 by intravaginal inoculation.